Molecular Biological Studies on Egg Cytoplasmic Factors That Are Responsible for Regulation of Gene Expression

推测早期海鞘胚胎的肌细胞具有极高的自主分化潜力。这种潜力归因于预定位的卵细胞质因子或决定因素。我们的项目的目的是了解因子的分子本质。解决这个问题的第一个方法包括分离和表征肌肉特异性基因和分析基因表达的调控因子。我们已经分离出肌球蛋白重链和肌肉型肌动蛋白的cDNA克隆。肌肉特异性基因的表达大约在原肠胚形成时开始，并且仅限于假定的肌肉细胞。研究表明海鞘胚胎合成几种不同的肌动蛋白mRNA。这些mRNA在其3 '非编码区的核苷酸序列上彼此不同，尽管编码区的序列几乎相同。其中一个克隆HrcMA 4含有1137 bp的开放阅读框和100 bp的非编码区，随后是poly（A）尾。利用以3 '端非编码区为主要成分的特异性探针，我们分离到了几个含有HrMA 4基因的基因组DNA片段。该基因的表征表明，HrMA4被两个内含子中断，它们的位置类似于脊椎动物骨骼肌肌动蛋白基因。另外，HrMA4和其他几个肌动蛋白基因形成了一个簇。第二种方法是表征肌浆的大分子，认为肌浆中含有细胞质因子。我们已经制备了几种特异性识别肌浆成分的单克隆抗体。其中一种抗体注射到受精卵后会阻止肌肉发育。抗原性多肽的表征揭示了该蛋白质与细胞骨架蛋白有关。

Presumptive muscle cells of early ascidian embryos have an extreme potential of autonomous differentiation. This potential is attributed to prelocalized egg cytoplasmic factors or determinants. The aim of our project is to understand the molecular nature of factors.The first approach to the problem includes an isolation and characterization of muscle-specific genes and analysis of regulatory factors for the gene expression. We have isolated cDNA clones for myosin-heavy chain and muscle-type actins. Expression of the muscle-specific genes begins around the time of gastrulation, and is restricted to presumptive muscle cells. It has been shown that the ascidian embryo synthesizes several different actin mRNAs. These mRNAs differ from one another in the nucleotide sequences of their 3'non-coding regions, although the sequences of the coding region are almost identical. One of the clones, HrcMA4 contains an open reading frame of 1137 bp and a 100 bp non-coding region followed by a poly (A) tail. Using a specific probe consisting mainly of 3'non-coding region, we have isolated several genomic DNA fragments containing HrMA4 gene. Characterization of the gene shows that HrMA4 is interrupted by two introns ; the position of them resembles to that of vertebrate skeletal muscle actin genes. In addition, it becomes clear that HrMA4 and several other muscle-actin genes form a cluster.The second approach is to characterize macromolecules of the myoplasm, which is thought to contain the cytoplasmic factors. We have made several monoclonal antibodies which specifically recognize the myoplasmic components. One of the antibodies blocks muscle development when it is injected into fertilized eggs. Characterization of the antigenic polypeptide has revealed that the protein is related to cytoskeletal proteins.